Proteins acquire their unique functions through specific folding of their linear polypeptide chains. Misfolding results in numerous diseases, such as cystic fibrosis and various neurodegenerative disorders. Although a great deal of work has been done on the investigation of how the secondary and tertiary structures of proteins are formed and both experimental and theoretical techniques for studying protein folding are continually becoming more refined, a quantitative and predictive understanding of protein folding is still not attainable. There are still many fundamental questions such as: How do specific and nonspecific interactions determine the folding pathways, the roughness of the energy landscape, the thermally and kinetically accessible conformation substates? On what range of timescales do particular conformational and folding events occur? Addressing these questions presents the need for further studies with static and time-resolved spectroscopic techniques that can provide the necessary time resolutions and structure sensitivities. The principal objective of the proposed research is therefore to develop new spectroscopic methods and new conformational probes that can be used to generate detailed structure interpretations of the stable and transient folding species and their dynamics over the time range of interest. A detailed set of experiments are planned to gain new insight into the understanding of various aspects of the protein folding problem. The technical goals are to: (a) develop a millisecond stopped-flow infrared spectrometer;(b) further develop new spectroscopic methods based on unnatural amino acids for protein folding and binding studies;(c) study the hidden intermediate in protein folding;(d) study the evolution of protein folding kinetics;(e) study the conformational and folding dynamics of single protein molecules. Achieving these specific aims should result in not only new experimental methods for protein folding and binding studies but also new insights into the understanding of many fundamental issues in protein folding, thus making practical and direct contributions to the field.